J. Org. Chem. 2001, 66, 4065-4068
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Frankiamide, a Highly Unusual Macrocycle Containing the Imide and Orthoamide Functionalities from the Symbiotic Actinomycete Frankia Karel D. Klika,† J. Pasi Haansuu,‡ Vladimir V. Ovcharenko,† Kielo K. Haahtela,‡ Pia M. Vuorela,§ and Kalevi Pihlaja*,† Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, Department of Biosciences, Division of General Microbiology, University of Helsinki, and Department of Pharmacy, Viikki Drug Discovery Technology Center, University of Helsinki, FIN-00014 Helsinki, Finland
[email protected] Received December 26, 2000
Frankia is a symbiotic actinomycete that forms nitrogenfixing root nodules in actinorhizal plants such as alder (Alnus sp.) and Casuarina sp., but it also commonly resides in soils lacking host plants.1-4 Recently, it has been shown that Frankiae frequently produce compounds with antimicrobial activity against Gram-positive Brevibacillus laterosporus and Gram-negative Pseudomonas solanacearum.5,6 These compounds may have a defensive role and, in addition to the iron-chelating siderophores, plant hormones, and hydrolyzing enzymes also produced by Frankiae, evidently assist Frankia, a slow growing microbe, to survive in nonsymbiotic conditions.7-11 Both Frankia strains G2 (ORS 020604) and ANP 190107 are also known to synthesize benzonaphthacene quinone metabolites that are structurally related to the antimicrobial compounds produced by some Streptomyces sp.,12-15 and two of these quinones were shown to possess biological activity by inhibiting the mitochondria of the yeast Candida lipolytica, the function of the respiratory chain in Paracoccus denitrificans, and the growth of Gram-positive Arthrobacter globiformis, the deuteriomycete Fusarium decemcellulare, and Candida lipolytica.16 Calcium channel antagonists, commonly used * Ph: 358-(2)-3336767. Fax: 358-(2)-3336750. † University of Turku. ‡ Department of Biosciences, Division of General Microbiology, University of Helsinki. § Department of Pharmacy, Viikki Drug Discovery Technology Center, University of Helsinki. (1) Huss-Danell, K.; Frej, A. K. Plant Soil 1986, 90, 407. (2) Maunuksela, L.; Zepp, K.; Koivula, T.; Zeyer, J.; Haahtela K.; Hahn, D. Microbiol. Ecol. 1998, 28, 11. (3) Smolander, A. Plant Soil 1990, 121, 1. (4) Smolander, A.; Ro¨nkko¨, R.; Nurmiaho-Lassila, E.-L.; Haahtela, K. Can. J. Microbiol. 1990, 36, 649. (5) Haansuu, P.; Vuorela, P.; Haahtela, K. Pharm. Pharmacol. Lett. 1999, 9, 1. (6) Lang, L. For. Res. 1999, 12, 47. (7) Arahou, M.; Diem, H. G.; Sasson, A. World J. Microbiol. Biotech. 1998, 14, 31. (8) Aronson, D. B.; Boyer, G. L. Soil Biol. Biochem. 1994, 26, 561. (9) Mansour, S. R.; El Melegy, S. A. Egypt. J. Microbiol. 1997, 32, 423. (10) Safo-Sampah, S.; Torrey, J. G. Plant Soil 1988, 112, 89. (11) Se´guin, A.; Lalonde, M. Plant Soil 1989, 118, 221. (12) Gerber, N. N.; Lechevalier, M. P. Can. J. Chem. 1984, 62, 2818. (13) Gomi, S.; Sasaki, T.; Itoh, J.; Sezaki, M. J. Antibiot. 1988, 41, 425. (14) Rickards, R. W. J. Antibiot. 1989, 42, 336. (15) Takeda, U.; Okada, T.; Takagi, M.; Gomi, S.; Itoh, J.; Sezaki, M.; Ito, M.; Miyadoh, S.; Shomura, T. J. Antibiot. 1988, 41, 417.
Figure 1. The structure of frankiamide (1) together with the numbering system in use. Note that the stereochemistry is unknown and is not inferred.
drugs for the treatment of cardiovascular disorders, have as their primary targets the slowly deactivating, lowactivation threshold voltage-sensitive calcium channels (VOCCs), and by their action Ca2+ influx is inhibited, resulting in the relaxation of vascular smooth muscle. Plant extracts containing phenolic compounds are known to affect the function of VOCCs,17 and several Frankia culture broth extracts have been shown to considerably inhibit Ca2+ fluxes in clonal rat pituitary GH4C1 cells.5 Recently, we isolated a particular Frankia strain, AiPs1, from a stand of Finnish Scots pine (Pinus sylvestris L.).2 After TLC fractionation of the microbial culture broth extract, only one antimicrobially active fraction was identified from which, using RP-TLC, was isolated a compound that inhibited the growth of several pathogenic fungi and Gram-positive bacteria. Moreover, this compound was also found to exhibit significant inhibition of 45 Ca2+ fluxes in clonal rat pituitary GH4C1 cells. The isolation procedure of the compound, together with the cultivation of the Frankia strain and assessment of the bioactivity of the isolated compound, are described in detail elsewhere,18 while this report describes the structure of the isolated compound (1), a novel macrocycle, which is depicted in Figure 1. To reflect both its origin and to allude to the inherent functionalities present within the system, we have dubbed 1 “frankiamide”. The structural elucidation of 1 only followed readily from NMR after some initial problems and the final realization of the molecular weight as 480 amu (not trivial, see below) and the resulting formula from HRMS analysis as C27H32N2O6. In methanol solution the compound exists as two readily interconverting forms (as evidenced by EXSY spectra) in the ratio of 3:1. That this interconversion was intricately linked to the concentration of Na+ ions was demonstrated by the addition of aqueous Na2HPO4 to the NMR sample, resulting in the shifting of the equilibrium further to one form. This dynamic process increased the complexity of the spectra, both by the presence of a greater number of signals and by the breadth of the peaks themselves, and made the (16) Medentsev, A. G.; Baskunov, B. P.; Stupar, O. S.; Nefedova, M. Y.; Akimenko V. K. Biokhimiya 1989, 54, 926. (17) Vuorela, H.; Vuorela, P.; To¨rnquist, K.; Alaranta, S. Phytomedicine 1997, 4, 167. (18) Haansuu, J. P.; Klika, K. D.; So¨derholm, P. P.; Ovcharenko, V. V.; Pihlaja, K.; Haahtela, K. K.; Vuorela, P. M. J. Ind. Microbiol. Biot. 2001, submitted for publication
10.1021/jo001789z CCC: $20.00 © 2001 American Chemical Society Published on Web 05/04/2001
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Notes
structural analysis more difficult. Although the addition of Na2HPO4 improved matters, it did not sufficiently alleviate the problem; neither did slowing the rate of exchange by lowering the temperature (-50 °C), and a sufficient increase in temperature to attain an averaged spectrum (>150 °C) only led to the rapid decomposition of the compound. In chloroform solution, however, this dynamic equilibrium was even more biased (>10:1). The interconversion was slower by comparison, and the resulting spectra were thus much more manageable; for this reason the NMR results are reported for CDCl3 solution. This behavior was also reflected in the HPLC analysis, which generally showed extremely broad peaks with front-end tailing, but fractions across the peak were assessed to be the same interconverting species by exchange spectra and by admixture, i.e., different fractions merely represented different positions of the equilibrium. Similarly, the chromatographic behavior could also be considerably improved by the addition of Na2HPO4 to the eluent. For the structural elucidation, gross sections of the molecule were readily apparent by NMR, such as the presence of a 2-substituted pyrrole ring (carbonyl adjacent to the ring), which was evident by the 1H chemical shifts and 1H coupling constants together with long-range 1 H-13C correlations. Mass spectral analysis also strongly inferred the presence of a carbonyl R to a pyrrole ring. The 1,2,3-trisubstituted phenyl ring (carbonyl adjacent to the ring at position 1, carbon at position 2, and oxygen at position 3) was evident by the 1H chemical shifts, 1H coupling constants, 13C chemical shifts, and long-range 1 H-13C correlations. The directly attached oxygen was proven to be a phenol group as a positive was obtained in a modified Folin-Ciocalteu phenol test19 (the other five oxygens were also discounted as bearing a proton, for three oxygens were present as carbonyls and the other two were each singly bound to two carbons). Finally, the alkyl segments proved to be all contained within one branched but unbroken alkyl chain. Tracing the alkyl chain from any distinct point (R to the carbonyls or the oxygen links) only led into the “alkyl forest”, and only methylene alkyl fragments were left unassigned, which were clearly only attached to other alkyl fragments by their chemical shifts, thus providing the deduction for the one alkyl chain. One of the carbonyls at the end of this chain was also shown to be the same carbonyl R to the pyrrole ring. Thus, what was remaining was an interesting quaternary carbon at 96.4 ppm and a tertiary nitrogen at -161.5 ppm; both were easily placed as a result of the observation of suitable long-range correlations. Since the long-range correlations, both 1H-{13C} and 1H-{15N} HMBC experiments and 13C-{1H} selective INEPT, played, as expected, such an instrumental role in placing these fragments together, the decisive correlations are depicted in Figure 2. By default, the quaternary carbon at 96.4 ppm and a tertiary nitrogen at -161.5 ppm must be bonded to one another and the same carbon (at 96.4 ppm) bound to the aromatic carbocycle. (No other combinations are sensible, and only one H was shown to be N-bound, the pyrrole H.) This remarkable connection, in addition to the fused ring systems, gives rise to both the imide and the orthoamide functionalities. Neither of these functional-
ities appears to have been previously reported as being present in a natural product. The mass spectra of 1 under EI+ conditions is consistent with the structure provided by NMR. The presence of the pyrrole-containing side chain is confirmed by the formation of stable C7H9NO+• (m/z 123) and C5H4NO+ (m/z 94) ions. This latter ion represents the base peak, and the formation of this acylium cation is quite characteristic for acyl pyrroles.20 Although the EI+ spectrum of compound 1 varies greatly with experimental conditions (e.g., heating rate, final probe temperature, etc.) and the molecular ion is not always observed, there are several consistent fragment peaks that appear even at moderate heating of the probe and are suitable for accurate mass measurements (see Table 1) and the determination of metastable transitions (fragmentation patterns are depicted in Figure 3). That the positive charge is mostly retained in the acyl pyrrole moiety accounts for the fragmentation of the polycyclic part of the molecule giving rise to only less prominent ions in the range of m/z 130-340. Of note though, consecutive losses of water (×3) and CO from the A fragment result in the formation of the fairly stable ion C19H17NO+• (m/z 275), in which the polycyclic structure is presumably largely preserved as only one carbon atom is lost from the A fragment (Figure 3). The polycyclic structure is, however, cleaved at the later stages of fragmentation with the formation of C10H12NO+ (m/z 162) and C9H5NO2+• (m/z 159) ions, which judging from their elemental compositions must be isoindolonerelated structures. What is remarkable about the EI+ spectra of 1 is the presence of adducts (clusters) between the molecule and its alkaline salt, [MSNa]+• and [MSK]+• at m/z of 982 and
(19) Nurmi, K.; Ossipov, V.; Haukioja, E.; Pihlaja, K. J. Chem. Ecol. 1996, 22, 2023.
(20) Porter, Q. N. Mass Spectrometry of Heterocyclic Compounds, 2nd ed.; Wiley: New York, 1985; p 541.
Figure 2. The decisive 1H-{13C} and 1H-{15N} long-range correlations leading to the structural elucidation of frankiamide. Also indicated are the chemical shifts of significant 13C and 15N nuclei. Table 1. Accurate Mass Measurements by EI+ m/z
rel int (%)
998
